tbuckley/cabot-search huggingface dataset:
P21 Tyrosine and serine STAT3 phosphorylation drives cutaneous squamous cell carcinoma in recessive dystrophic epidermolysis bullosa as demonstrated using a 3D <i>in vitro</i> model
Authors: Virginia Llopis-Hernandez, Katarzyna Balon, Dawei Chen, Jemima Mellerio, John McGrath, Joanna Jacków
Journal: British Journal of Dermatology (2023)

Abstract The early onset of aggressive cutaneous squamous cell carcinoma (cSCC) and its rapid progression in recessive dystrophic epidermolysis bullosa (RDEB) leads to nearly 100% mortality rates in these patients. Elevated levels of phospho-STAT3 on tyrosine residue (STAT3Tyr705) have been intensively demonstrated in RDEB-derived cSCC cells, demonstrating that the constitutive activation and dysregulation of the Janus kinase (JAK)/STAT pathway plays a role in RDEB cSCC pathogenesis. We efficiently applied ruxolitinib (JAK1/2 inhibitor that blocks phosphorylation on STAT3Tyr705) to a RDEB–cSCC xenograph mouse model, reducing the size of small tumours but found that it was insufficient to reduce bigger tumours completely. Recently, phosphorylation at serine-727 (STAT3Ser727) has been shown to be relevant in the evolution of some cancers, but it has not yet been studied in RDEB cSCC. We hypothesized that phosphorylation at serine-727 of the STAT3 pathway might play a role in RDEB cSCC tumour development and progression, and could not be inhibited by ruxolitinib. To validate our hypothesis, we studied two RDEB cSCC human patient samples with different degrees of dedifferentiation (normal RDEB skin, edge of the tumour and centre of the RDEB cSCC). We demonstrated that STAT3Tyr705 was preferentially upregulated in the normal RDEB skin followed by the tumour edge skin, presenting less phosphorylation on the tumour. When we analysed STAT3 phosphorylation on STAT3Ser727, we could see that it presented the opposite phosphorylation pattern, being more phosphorylated in the tumour and less in normal RDEB skin. Furthermore, we validated the STAT3Ser727residue pattern across other cSCC cell lines and found that serine phosphorylation is present in other SCCs, demonstrating its crucial role in tumour development. To further investigate and analyse cancer progression in more detail, an easy and robust RDEB cSCC model is instrumental. Therefore, we generated RDEB cSCC spheroids in vitro, which has four key advantages over the previous models developed: (i) it is easy to generate; (ii) small amounts of cells are required; (iii) it is suitable for drug testing; and (iv) it can be used to recapitulate different stages of tumour development. We have been able to control their size and use them for drug testing, observing a 20% size decrease after 6 days of ruxolitinib treatment. Taken together, this three-dimensional RDEB cSCC in vitro model, along with analysis of human samples, will allow us to better understand RDEB cSCC progression at different stages, allowing us to investigate new treatments for RDEB cSCC.
O11 The human discs large protein (Dlg1) controls connexin 43 (Cx43) plasma membrane localization and gap junctional communication in keratinocytes
Authors: Harry Scott, Li Dong, Sharmila Srinivasan, Andrew Stevenson, Alasdair MacDonald, Paola  Massimi, Lawrence Banks, Patricia Martin, Scott Johnstone, Sheila Graham
Journal: British Journal of Dermatology (2023)

Abstract Gap junctional communication is required for cellular coordination in health and disease. Gap junction channels are composed of connexins and allow direct intercellular ­communication. Connexins also play an important role in processes such as wound healing, with connexin 43 (Cx43) being downregulated at the plasma membrane of cells at the wound edge during the initial stages of the normal wound healing process. Connexins must be located on the plasma membrane in order to form gap junctions; therefore, it is important to identify proteins involved in the regulation of this. Previously, in a study of human papillomavirus (HPV)-positive tumour cells, we reported that the gap junction protein Cx43 is a physiologically relevant binding partner of the human homologue of Drosophila Discs large (Dlg1). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family that is known to control cell shape and polarity, and intracellular trafficking. Here we show, for the first time, that Cx43 interacts with Dlg1 in uninfected keratinocytes in vitro and in normal human epithelial tissues. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with an overall reduction in Cx43 protein levels. The loss of Dlg1 in keratinocytes was further associated with a loss of Cx43 at the plasma membrane and a reduction in intercellular communication. This decrease in Cx43 levels was partially reversed through use of a lysosomal inhibitor; however, a proteasomal inhibitor resulted in no difference to Cx43 levels, suggesting that, in the absence of Dlg1, Cx43 is degraded through the lysosomal but not the proteasomal pathway. This correlated with an increased association between Cx43 and LAMP2, a marker of lysosomes in Dlg1 knockdown cells. In the absence of Dlg1, Cx43 localized to the Golgi but not the endoplasmic reticulum. Although this result could indicate that Dlg1 is required for stable Cx43 membrane location, a study of Cx43 and Dlg1 during the wound healing process in HaCaT cells (a spontaneously immortalized HPV-negative epithelial cell line), showed the proteins co-localizing as they relocated from the plasma membrane to the cytoplasm between 4 and 16 h postwounding before returning to the plasma membrane at 24 h postwounding. This may suggest that Dlg1 is involved in Cx43 trafficking. The levels of both proteins were reduced between 0 and 4 h postwounding and then increased between 4 and 16 h, before returning to normal levels at 24 h postwounding. Taken together, our data suggest that Dlg1 is crucial for promoting plasma membrane localization of Cx43 in keratinocytes.
O14 Junctional epidermolysis bullosa: repairing the epidermal lipid barrier
Authors: Eleri Jones, Supatra Marsh, Ryan O'Shaughnessy, Monique Aumailley, John  McGrath, Edel  O’Toole, Matthew Caley
Journal: British Journal of Dermatology (2023)

Abstract Junctional epidermolysis bullosa (JEB) caused by loss of function mutations in basement membrane genes, including that encoding laminin-332, is among the most severe forms of epidermolysis bullosa. Affected individuals suffer from blistering from birth, leading to scarring, granulation tissue and susceptibility to infection. In generalized severe JEB, there is a 50% mortality rate in the first 2 years of life, often due to failure to thrive or overwhelming infection. Using cells with knockdown of laminin-α3 we generated a RNAseq data set and have identified a new pathway altered in JEB, the regulation of cholesterol biosynthesis. It has been shown that loss of laminin-332 results in reduced epidermal lipid barrier specifically due to loss of cholesterol transport within keratinocytes. Cholesterol biosynthesis and cholesterol trafficking are fundamental processes in a wide range of cells and tissues, especially in the formation of the normal epidermal lipid barrier. A cholesterol transport screening assay has been developed and used to analyse the ability of 10 specifically selected compounds to restore cholesterol transport in our in vitro model of JEB. Results of this small-scale screen showed that nine of 10 of the selected drugs were able to restore cholesterol transport up to 50% of control. Six drugs were selected for further analysis within our three-dimensional (3D) skin-equivalent model of JEB. Organotypic skin equivalents of normal skin (siControl) and JEB skin (siLaminin-α3) were treated daily for 7 days with either dimethyl sulfoxide or 1 µmol L–1 of selected drugs. In 3D, drugs 1, 3, 5 and 6 displayed an increase in stratum corneum lipids shown by Nile red staining. Drugs 4 and 7 did not increase stratum corneum lipids above levels seen in the untreated JEB organotypics. Further analysis of skin differentiation by keratin, involucrin and tranglutaminase staining were assessed to narrow drug selection for further testing. These data also suggest that JEB is not just a disorder of epidermal–dermal adhesion, but a disorder of aberrant cholesterol trafficking causing a markedly impaired skin barrier. These findings suggests that drugs to target cholesterol transport within keratinocytes could be a beneficial treatment for JEB, helping to improve the crucial epidermal lipid barrier and prevent water loss and infection at the early stages of life.
P10 Inducible 14-3-3 sigma/stratifin ablation cooperates with rasHa activation to accelerate papillomatogenesis and induce malignant conversion in transgenic mouse skin carcinogenesis
Authors: Rebecca Hardman-Carter, Revvekka Lefkati, Carol  McMenemy, David Greenhalgh
Journal: British Journal of Dermatology (2023)

Abstract In carcinogenesis, the 14-3-3σ isoform (stratifin) is implicated by being downregulated in a mode of classic tumour suppression gene (TSG) loss and also via elevated oncogenic roles; with both scenarios appearing to be hallmarks of 14-3-3σ deregulation, depending on tumour stage or context. Mouse skin models highlight tumour-suppressive roles, as the Er/Er+/– repeated epilation stain possesses germline mutations that result in hyperplasia, failed follicular differentiation and susceptibility to squamous cell carcinoma (SCC). In transgenic models, 14-3-3σ knockout supports early roles, resulting in rapid papilloma formation in classic two-stage DMBA/TPA chemical carcinogenesis; however, our previous studies in this model demonstrated targeted overexpression of 14-3-3σ led to SCC of a follicular origin in collaboration with Fos and well-differentiated SCC in collaboration with rasHa. Therefore, to study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of endogenous 14-3-3σ (stratifin) was analysed in early skin carcinogenesis driven by activated rasHa expression (HK1.ras) and subsequently via conditional RU486-inducible 14-3-3σ knockout (K14.creP-Δ14-3-3σ). Consistent with 14-3-3σ roles previously observed in epidermal differentiation, HK1.ras hyperplasia and early papillomas displayed elevated 14-3-3σ expression in suprabasal keratinocytes and in occasional K1-positive basal keratinocytes as they committed to differentiate. Older HK1.ras mice exhibited increasing 14-3-3σ positivity in papilloma basal layers, suggesting attempts to protect basal-layer p53 TSG function, given that 14-3-3σ acts as a chaperone protein to remove/relocate MDM2 for degradation, thus maintaining p53 levels. Indeed, such papillomas expressed activated p-MDM2166 confined to suprabasal layers, resulting in strong basal layer p53 expression and thus HK1.ras papillomas lacked malignant conversion. In mice where 14-3-3σ functions were ablated via an RU486-inducible Cre/loxP system expressed from a keratin 14 (K14) promoter (K14.creP-Δ14-3-3σ), treated HK1.ras-K14.Δ14-3-3σ mice exhibited a rapid papillomatogenesis and produced larger papillomas. These papillomas also exhibited rapid conversion to malignancy, associated with increased activated p-MDM2166 expression in basal layers and resultant p53 loss. These observations suggest that 14-3-3σ plays tumour suppressive roles involving p53 protection, where failure to regulate MDM2 activity led to p53 loss and susceptibility to malignant conversion. P11 Abstract withdrawn.
O18 Molecular crosstalk between PLCγ1 and STAT3 in cutaneous T-cell lymphoma
Authors: Victoria Campbell, Charlotte Flanagan, Isabella  Tosi, Christine Jones, Sean Whittaker, Susan John, Tracey Mitchell
Journal: British Journal of Dermatology (2023)

Abstract Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes that is difficult to diagnose and treat due to a high degree of clinical and genomic heterogeneity. Mutations in PLCG1/PLCγ1 occur in 10% of CTCL tumours. Meanwhile, signal transducer and activator of transcription 3 (STAT3) – a common driver of lymphomagenesis – is frequently activated (pY705-STAT3) in CTCL in the absence of mutation. Activation of STAT3 may also be mediated by serine phosphorylation (pS727-STAT3), and there is a lack of data on the role of this modification in CTCL. We aimed to identify if gain-of-function PLCG1 mutations in CTCL activate STAT3, either directly or indirectly, via downstream mitogen-activated protein kinase (MAPK) signalling. Flow cytometry was used to study STAT3 phosphorylation and subcellular localization in ex vivo CD4+-enriched tumour cell populations from patients with CTCL and healthy donors. STAT3 phosphorylation was also studied in the J.gamma1 T-cell line (PLCγ1-null) and primary ex vivo CD4+ cells upon lentiviral overexpression of wild-type (WT) and mutant (p.R48W, p.S345F) PLCγ1 constructs. To investigate the role of MAPK signalling, the CTCL-derived Hut78 and SeAx cell lines were treated with MAPK inhibitors (trametinib), and STAT3 phosphorylation assessed by Western blotting. Significantly increased basal levels of pS727-STAT3 (P &amp;lt; 0.05) and pY705-STAT3 (P &amp;lt; 0.001) were observed in CD4+-enriched populations from patients with Sézary syndrome compared with healthy donors. Mitochondrial translocation of STAT3 was also increased in CD4+-enriched populations from patients with Sézary syndrome (P &amp;lt; 0.05). In transduced J.gamma1 cells, p.S345F-PLCγ1 overexpression activated pS727-STAT3 (P &amp;lt; 0.05), while overexpression of both WT and mutant PLCγ1 constructs activated pS727-STAT3 in primary ex vivo CD4+ cells (P &amp;lt; 0.05 and P &amp;lt; 0.01, respectively). Inhibition of MAPK kinase (MEK)–extracellular signal regulated kinase (ERK) signalling did not significantly modulate STAT3 phosphorylation or subcellular localization in CTCL cell lines, suggesting that pS727-STAT3 phosphorylation is not mediated via ERK in CTCL. This work extends our understanding of PLCγ1 signalling and identifies pS727-STAT3 as a potential target of interest for further study in CTCL.
O03 HPV8 leads to Lrig1+ keratinocyte stem cell expansion into the overlying epidermis
Authors: Huw J Morgan, Boris Shorning, Carlotta Olivero, Cinzia Borgogna, Baki Akgul, Marisa Gariglio, Girish K Patel
Journal: British Journal of Dermatology (2023)

Abstract Actinic keratoses (AKs) represent the precursor skin lesions in the pathogenesis of up to 87% of cutaneous squamous cell carcinomas (cSCCs). However, current treatment of AK lesions, at best, lead to only a 25% reduction in cSCC and relies on therapies that target abnormal keratinocytes using topical chemotherapy, immune stimulants or surgical resection. We have previously shown that 44% of AK tissue sample histologies contain koilocytes, wherein genotyping universally identified human papillomavirus 8 (HPV8). To determine the role of HPV8 in the pathogenesis of AKs, we used the K14–HPV8 complete early region (HPV8tg) to generate triple cross-lineage tracing transgenic mice: HPV8:K5-CreERT2:R26R-LoxP-flanked-Brainbow2.1 (Confetti reporter); HPV8:Lrig1-CreERT2:R26R-LoxP-flanked-Brainbow2.1; and HPV8:K15-PGR:R26R-LoxP-flanked-Brainbow2.1. Upon Cre activation, progeny clones exhibit one of four fluorescent colours. Compared to wild-type mice, all HPV8tg mice exhibited an expansion of keratinocytes in the hair follicle infundibulum (77.09 mm3 and 253.43 ± 54.15 mm3) and overlying epidermis. Clones from the Lrig1, but not K15, keratinocyte stem cell population were responsible for keratinocyte expansion of both the hair follicle infundibulum and overlying epidermis. Compared with wild-type mice, HPV8tg mice exhibited greater colony-forming efficiency (1.75 ± 0.69% vs. 6.27 ± 2.41%; n = 3), and, consistent with keratinocyte stem cell expansion, ΔNp63 expression was increased 4.14 ± 2.10-fold (P &amp;lt; 0.05, n = 5). Flow-sorted epidermal clones from wild-type and HPV8tg mice were examined by RNA sequencing (n = 5). Warthin–Starry staining showed that the expanded population had no melanin ‘cap’ in our mouse models and human AK samples. In summary, expression of HPV8 early-region genes is associated with AK, wherein there is an expansion of the Lrig1, but not K15, keratinocyte stem cell population that results in epidermal thickening reminiscent of human AK pathology. These findings suggest that HPV8 results in translocation of hair follicle keratinocyte stem cells into the interfollicular epidermis without melanin production, making these cells susceptible to ultraviolet light transformation.
O05 Functional roles of lncRNA in cutaneous squamous cell carcinoma disease progression
Authors: Max Bone, Catherine Harwood, Irene Leigh, Peter Bailey, Gareth Inman
Journal: British Journal of Dermatology (2023)

Abstract Cutaneous squamous cell carcinoma (cSCC) is one the most prevalent malignancies with metastatic potential within the UK and is of significant public concern due to its increasing annual incidence. The progression of cSCC has been historically characterized into several key stages, but the mechanistic properties as to how the disease advances remains poorly defined. In recent years, long noncoding RNA (lncRNA), a widely uncharacterized and poorly understood family of RNA molecules over 200 nucleotides in length, have become of increasing interest in regulating epigenetics and various other cellular processes. These molecules have also been found to be widely associated with cancer pathways as both inhibitors and drivers of disease, and have been identified in all cancers as potential molecules of interest, including cSCC. Although several lncRNA have been recognized as functionally relevant in cSCC, they represent a small proportion of the estimated 300 000 lncRNA transcripts of which most have unknown functions within both skin and cSCC specifically. By performing RNAseq on 110 patient samples, including normal sun-exposed skin (n = 26), actinic keratosis (n = 14), primary cSCC (n = 66) and metastases (n = 4) and utilizing various bioinformatic analysis pipelines, key lncRNA of interest were identified in cSCC. These lncRNA of interest were then explored in vitro in a panel of patient-derived cSCC cell lines to find key lncRNA of functional relevance. These functionally active lncRNA were subjected to further in silico analysis to determine splice isoforms and transcripts responsible for disease progression, and potential mechanisms of regulating cellular epigenetics.
P20 Engineered peptide for KLK5 inhibition – therapeutic intervention for atopic dermatitis
Authors: Jeffrey Mah, Derek Macmillan, Wei-Li Di
Journal: British Journal of Dermatology (2023)

Abstract Epidermal proteases, such as human tissue kallikrein-related peptidase, regulate skin barrier function through keratinocyte desquamation, antimicrobial defence, inflammatory response and barrier maintenance. It has been reported that the activity of kallikreins (KLKs) is upregulated in inflammatory skin disorders, including atopic dermatitis (AD; eczema). AD is a very common, chronic and relapsing inflammatory skin disease affecting 15–20% of children in the UK. Studies have shown that enhanced KLK5 results in AD-like skin architecture with an impaired skin barrier function. In this study, we aimed to develop a potent and selective inhibitor that targets KLK5 and restores epidermal barrier dysfunction in AD. Nature-based sunflower trypsin inhibitor 1 (SFTI), a small, circular peptide exhibiting wide-ranging serine protease inhibition can be engineered and modified to become a potent inhibitor specific for tissue kallikreins; our previous study also indicated that it was able to permeate through the outermost layer to exert its inhibitory action on serine proteases. In this study, we wanted to develop a more potent and specific inhibitor of KLK5 based on a lead SFTI analogue (‘analogue 6’) that was previously developed by our research group. Computer modelling was employed to assess the structure and binding affinity between KLK5 and SFTI-1. Designed SFTI analogues were then synthesized using solid phase peptide synthesis. The inhibitory effect was evaluated by its half maximal inhibitory concentration (IC50) against KLK5 using a 7-amido-4-methylcoumarin assay and further tested by protease-activated receptor 2-dependent intracellular calcium influx assay in keratinocytes. Computer modelling then indicated that an endogenous inhibitor was comparable with the analogue 6 we developed previously. Analogue 6 was further modified, based on the endogenous inhibitor structure, and synthesized. The new analogue achieved an IC50 of 14 nmol L–1 for KLK5, which was a fourfold increase in potency compared to analogue 6 (IC50 56 nmol L–1) and a 16-fold increase vs. SFTI-1 wild type (IC50 230 nmol L–1). Further, the new analogue could inhibit proteinase-activated receptor 2 (PAR2)-dependent intracellular calcium influx by modulating the activation of PAR2 receptor, a KLK5 downstream molecule in keratinocytes at a minimum inhibitor-to-enzyme molar ratio of 1:1. The novel potent inhibitor for KLK5 could be a potential therapeutic intervention for AD and other inflammatory skin conditions with enhanced KLK5.
P26 Characterization of skin barrier defects in atopic dermatitis patients using attenuated total reflection Fourier transform infrared spectroscopy
Authors: Samuel Williams, Simon Danby
Journal: British Journal of Dermatology (2023)

Abstract Atopic dermatitis (AD) is a common disorder characterized by skin barrier defects often measured by traditional biophysical tools that give little molecular insight into disease pathogenesis. The objective was to employ in vivo infrared spectroscopy as a complimentary technique to biophysical measurements to analyse changes in the chemical composition of the stratum corneum (SC) in relation to AD severity. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy measurements were collected on the forearm alongside transepidermal water loss (TEWL), skin surface pH and capacitance in a cohort of 75 participants, stratified by AD severity. Skin tape stripping was performed to allow measurements at greater SC depth. The five common filaggrin (FLG) loss-of-function mutations were genotyped. Skin hydration, TEWL and redness were all affected by AD severity with molecular analysis showing concomitant changes in lipid structure, and the amount of lipid, polyamide and carboxylate. Correlation analysis showed associations between Eczema Area and Severity Index and lipid structure (r = –0.58), as well as amount of carboxylate (r = –0.49). Principal component analysis showed spectra collected on participants with AD lesions or FLG mutations clustered away from healthy and nonlesional skin sites. Our findings showed that ATR-FTIR spectroscopy is able to characterize structural skin barrier defects in people with AD vs. controls. Spectral peak analysis showed associations between defective lipid and natural moisturizing factor (NMF) components with worsening disease severity. Through combined assessment of peaks associated with lipid structure and carboxylate amount, we have shown the potential of ATR-FTIR in identifying filaggrin deficiency, through either genetic predisposition (FLG mutations) or through inflammatory action, which is related to the presence of NMFs in the SC, skin hydration and overall severity of AD.